Project Summary Cholera is a severe dehydrating diarrheal disease caused by Vibrio cholerae, a Gram-negative rod that colonizes the small intestine and produces cholera toxin, whose actions largely account for the symptoms of cholera. Prevention of cholera through utilization of an effective vaccine could be of enormous benefit. However, the currently available vaccine, Shancol, a killed whole cell preparation, has several limitations including only modest efficacy, requirement for two doses, and a duration of activity that is likely less than that engendered by natural infection with wild type V. cholerae. Live-attenuated vaccines offer the possibility of overcoming these limitations, but the last live-attenuated vaccine for cholera that showed promise in human trials was created more than 25 years ago and is no longer being developed. In the interim, the V. cholerae strain causing cholera worldwide has evolved. A `variant' El Tor strain, the cause of the devastating and ongoing cholera outbreak in Haiti, is now the predominant cause of cholera globally. During the current grant period, we used the infant rabbit-based model of cholera we developed to create new approaches for deepening our understanding of V. cholerae-host interactions during infection and created a new live- attenuated vaccine, HaitiV, the first created in the variant El Tor V. cholerae background. Infant rabbits orogastrically infected with HaitiV are colonized, but do not develop diarrhea, due to the absence of cholera toxin and other diarrheagenic factors. Moreover, administration of HaitiV reduces subsequent intestinal colonization by and diarrhea associated with wild type V. cholerae even prior to the development of adaptive immunity. Killed HaitiV does not induce acute colonization resistance or protection from disease, suggesting that killed vaccines (e.g. Shancol) would likewise lack this benefit. Studies to address HaitiV immunogenicity are outside the scope of this proposal, but we anticipate, based on prior clinical studies of live vaccines, that HaitiV will induce long-term immunity to cholera after a single dose. Rapid HaitiV vaccination-induced colonization resistance also has great potential clinical impact and is amenable to study in infant rabbits. In Aims 1 and 2 of this proposal, we will investigate how HaitiV antagonizes intestinal colonization by wild type V. cholerae. In Aim 3, we will test whether we can augment HaitiV's capacity to prevent intestinal colonization and disease caused by wild type V. cholerae. Collectively, these experiments will broaden our understanding of the processes that govern V. cholerae intestinal colonization and colonization resistance. In addition, these studies will potentially yield a transformative, multifunctional agent that will be effective both as a traditional vaccine, inducing long term immunity, as well as for reactive vaccination for controlling the acute spread of epidemics even before stimulation of an adaptive, long-lived immune response.